These studies, all derived from our previous work exploring the interactions of bone matrix and bone resorption, focus on genes and other factors impacting skeletal development and resorption. Specific Aim 1 proposes mapping two unique mutations responsible for the failure of osteoclast development and function in the rat. This aim will be pursued using classical genetics combined with molecular mapping and positional cloning techniques to identify the genes responsible for the osteopetrotic phenotypes of the incisor absent (ia) and toothless (tl) rat mutations. These inbred strains that we have maintained for many years will be outcrossed with a highly polymorphic strain. A series of PCR reactions, linkage studies, and ultimately detailed sequence analyses will be used to identify the mutated genes. Candidates from a newly identified region in the tl rat are currently being evaluated. Specific Aim 2 explores the functional relationship between connective tissue growth factor (CTGF) secretion by osteoblasts and osteoclast recruitment and activation. We have shown up-regulation of CTGF mRNA and protein in vivo by osteoblasts in three osteopetrotic mutations in the rat and one in the mouse, all of which intercept osteoclast differentiation and/or function at distinct points. The commonality of CTGF over-expression implies that bone resorption is required to reduce CTGF to normal levels. We will test this hypothesis: (1) by curing mutant animals and following the impact on CTGF levels in vivo; (2) using osteoclast and bone organ culture systems, CTGF/osteoclast binding will be prevented by anti-CTGF antibodies and by saturating osteoclast alpha(v)beta(3) integrins with recombinant CTGF, and the effect on resorption will measured; (3) supernatants from resorbing cultures will be tested for their ability to down-regulate CTGF expression by osteoblasts. Specific Aim 3 will establish whether collagen gene switching dis-regulation is a common feature of growth abnormalities at craniofacial synchondroses and sutures. We have shown failures of collagen gene switching (types I and Ill and types II and X) at two key growth centers in the tl rat. We will extend these studies to the synchondroses of the skull base, and use this approach to screen a series of mice with craniofacial and/or osteopetrotic mutations for similar failures. This will establish how frequently collagen expression abnormalities accompany craniofacial defects, whether it constitutes a potential "read-out" for craniofacial growth defects, and therefore whether mechanistic future investigations are warranted. Hypothesis/Specific Aim 4. Extending studies of abnormal gene expression in osteopetrotic mutations will uncover more genes involved in regulating normal skeletal metabolism. We have previously used differential display of mRNA to discover several important skeletal genes. We will extend these studies using high-density gene array technology to compare normal, osteopetrotic, and cured osteopetrotic bone RNA to identify more genes and pathways that are up- or down-regulated in osteopetrotic mutations and that underlie osteoclast differentiation in vivo. This method will also permit us to follow the persistence of abnormal expression or its return to normal following restoration of resorption by cytokine treatments.